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Functional expression of Candida antarctica lipase B in Eschericha coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
25587 43583 2006 10 PDF Available
Title
Functional expression of Candida antarctica lipase B in Eschericha coli
Abstract

Candida antarctica lipase B (CalB) is an important catalyst in bio-organic synthesis. To optimize its performance, either the reaction medium is changed or the lipase itself is modified. In the latter case, mutants are generated in Eschericha coli and subsequently expressed in fungal hosts for their characterization. Here we present the functional expression of CalB in the periplasm of E. coli. By step-wise deletion of the CalB signal and propeptide we were able to express and purify two different variants of CalB (mature CalB and CalB with its propeptide). A N-terminal FLAG and a C-terminal His tag were used for the purification. For the substrates para-nitrophenol butyrate (p-NPB), para-nitrophenol laurate (p-NPL) and carboxyfluorescein diacetate (CFDA) the specific activity was shown to be similar to CalB expressed in Aspergillus oryzae. The kinetic constants kM, vmaxvmax and kcat were determined using the substrates p-NPB and p-NPL. Almost identical kcat/kM values (0.423–0.466 min−1 μM−1 for p-NPB and 0.068–0.071 min−1 μM−1 for p-NPL) were obtained for the CalB variants from E. coli and A. oryzae. The results clearly show that CalB can be functionally expressed in E. coli and that the attachment of tags does not alter the properties of the lipase.

Keywords
CalB; Candida antarctica; Lipase activity; Periplasmic expression; Propeptide
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Functional expression of Candida antarctica lipase B in Eschericha coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 125, Issue 4, 1 October 2006, Pages 474–483
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us